Identification of Fluorescent Compounds with Non-Specific Binding Property via High Throughput Live Cell Microscopy

TitleIdentification of Fluorescent Compounds with Non-Specific Binding Property via High Throughput Live Cell Microscopy
Publication TypeJournal Article
Year of Publication2012
AuthorsNath S, Spencer VA, Han J, Chang H, Zhang K, Fontenay GV, Anderson C, Hyman JM, Nilsen-Hamilton M, Chang Y-T, Parvin B
Journal TitlePLoS ONE
Volume7
Pagese28802
Date Published01
Abstract

Introduction

Compounds exhibiting low non-specific intracellular binding or non-stickiness are concomitant with rapid clearing and in high demand for live-cell imaging assays because they allow for intracellular receptor localization with a high signal/noise ratio. The non-stickiness property is particularly important for imaging intracellular receptors due to the equilibria involved.

Method

Three mammalian cell lines with diverse genetic backgrounds were used to screen a combinatorial fluorescence library via high throughput live cell microscopy for potential ligands with high in- and out-flux properties. The binding properties of ligands identified from the first screen were subsequently validated on plant root hair. A correlative analysis was then performed between each ligand and its corresponding physiochemical and structural properties.

Results

The non-stickiness property of each ligand was quantified as a function of the temporal uptake and retention on a cell-by-cell basis. Our data shows that (i) mammalian systems can serve as a pre-screening tool for complex plant species that are not amenable to high-throughput imaging; (ii) retention and spatial localization of chemical compounds vary within and between each cell line; and (iii) the structural similarities of compounds can infer their non-specific binding properties.

Conclusion

We have validated a protocol for identifying chemical compounds with non-specific binding properties that is testable across diverse species. Further analysis reveals an overlap between the non-stickiness property and the structural similarity of compounds. The net result is a more robust screening assay for identifying desirable ligands that can be used to monitor intracellular localization. Several new applications of the screening protocol and results are also presented.

URLhttp://dx.doi.org/10.1371%2Fjournal.pone.0028802
DOI10.1371/journal.pone.0028802